Prior to phenotypic NAI sensitivity testing, fecal samples were propagated in SPF ECE (Valo, Germany), as described above, to obtain a sufficient viral titer for the assay, followed by NA sequencing (as described above) to verify the genotype.
Total RNA was collected and analyzed as described above to verify these knockdowns at 24 hours post-transfection, with cell growth assessed 72 hours post-transfection by CellTiter-Glo assay, as already described.
For these reasons, we chose to perform multiple techniques described below to verify the role of Sp1 in M-CSF-induced VEGF production from these cells.
We also used the in vitro model (described above) to verify the therapeutic efficacy of salubrinal (a selective inhibitor of eIF2α de-phosphorylation) in controlling protein folding and processing based on its ability to reduce the accumulation of ubiquitinated proteins.
After incubation, the cells were rinsed 3 times with fresh media and both bright field and fluorescent images were acquired using the same microscope set-up described above to verify that the lysosomes and endosomes were efficiently labeled (Figure S2).
Early reading skills were estimated with the tasks described below to verify that children were showing age appropriate progress on those skills (based on the norms provided by the test creators) and to allow us to compare the relationships between frequency discrimination, verbal memory and phonological awareness to those previously reported in adolescents [5], [24].
Additional peptide sequencing was done by MALDI TOF/TOF mass spectrometry (described above) to verify protein identification.
Colony PCRs were performed as described above to verify the successful cloning by using vector-specific primers.
Using the interrogation methods described above to verify adduction sites, two lysine residues, K190 and K351, displayed the highest reactivity (Table 1).
One fifth of this sample was analysed by MALDI-TOF MS as described above, to verify the presence of peaks of interest.
